The invention relates to methods of diagnosis for Alzheimer's Disease in humans. More particularly, the present invention is a diagnostic assay based on the detection of proteolysis of the precursor to the Alzheimer's Disease beta-amyloid protein in the presence of a sample of cerebrospinal fluid or blood obtained from a patient to be tested.
Alzheimer's Disease (hereinafter "AD") is a progressive, degenerative disorder of the brain, characterized by progressive atrophy, usually in the frontal, parietal and occipital cortices. The clinical manifestations of AD include progressive memory impairments, loss of language and visuospatial skills, and behavioral deficits (McKhan et al., 1986, Neurology 34:939-944). Overall cognitive impairment is attributed to degeneration of neuronal cells located throughout the cerebral hemispheres (Price, 1986, Annu. Rev. Neurosci. 9:489-5120).
Pathologically, the primary distinguishing features of the post-mortem brain of an AD patient are, 1) pathological lesions comprised of neuronal perikarya containing accumulations of neurofibrillary tangles; 2) cerebrovascular amyloid deposits; and 3) neuritic plaques. Both the cerebrovascular amyloid and the neuritic plaques contain a distinctive peptide simply designated, "A4" or "beta-amyloid".
Beta-amyloid is an insoluble, highly aggregating, small polypeptide of relative molecular mass 4,500, and is composed of 39 to 42 amino acids. Kang et al., 1987, Nature 325:733-736, described the beta-amyloid protein as originating from and as a part of a larger precursor protein. To identify this precursor, a full-length complementary DNA clone coding for the protein was isolated and sequenced, using oligonucleotide probes designed from the known beta-amyloid sequence. The predicted precursor contained 695 residues and is currently designated, "APP 695" (amyloid precursor protein 695).
APP 695 is the most abundant form of APP found in the human brain, but three other forms exist, APP 714, APP 751 and APP 770. The different length isoforms arise from alternative splicing from a single APP gene located on human chromosome 21 (Goldgaber et al., 1987, Science 235:877-880; Tanzi et al., 1987, Science 235:880-885).
Subsequent cloning of the gene encoding the APP proteins revealed that the A4 region was encoded on two adjacent exons, ruling out the possibility that A4 accumulation is the result of direct expression of an alternatively spliced mRNA. This implied that A4 accumulation must result from abnormal proteolytic degradation of the APP at sites both N- and C-terminal to the peptide region within the APP.
Recent studies have shown that APP fragments extending from the N-terminus of A4 to the C-terminus of the full length APP molecule (referred to hereinafter as the "C-100 fragment", because it is comprised of approximately 100 amino acids) are also capable of aggregation (Wolf et al., 1990, EMBO 9:2079-2084). Furthermore, overexpression of C-100 fragments in transgenic mice results in the accumulation of neurofibrillary tangles and neuritic plaque co-incident with neuronal degeneration (Kawabata et al., 1991, Nature 325:476-478). Collectively these data suggest that a single proteolytic cleavage of APP at the N-terminus of the A4 region is sufficient to initiate the pathophysiology associated with AD.
APP is also cleaved at a site within the A4 region in the physiological pathway for secretion of the APP extracellular domain (Esch et al., 1990, Science 2:1122-1124; Wang et al., 1991, J. Biol. Chem. 266:16960-16964). This pathway is operative in several cell lines and necessarily results in the destruction of the A4, amyloidic region of the precursor. Evidence that such a pathway is also operative in the human brain has been obtained (Palmert et al, 1989, Biochem. Biophys. Res. Comm. 165:182-188).
The enzymes responsible for the normal, non-pathological processing of APP have been termed "secretases". C-terminal fragments resulting from secretase action are smaller than the C-100 fragments (defined above) by 17 amino acids, and will hereinafter be referred to as the "physiological C-terminal fragment."
It has been postulated that the net pathological accumulation of A4 is controlled by the relative activity of the pathologic and physiologic pathways of APP degradation. It is uncertain whether the imbalance resulting in the dramatic increase in accumulation of A4 in the brains of AD patients results from a decrease in the activity of the secretases or an increase in the pathologic protease activity, or a combination of both.
Several studies have undertaken the purification and characterization of both the secretases and purported pathologic proteases. Initial studies utilized assays featuring synthetic peptide substrates that only mimicked the expected cleavage sites within APP. These assays failed to provide the necessary protease specificity, and the peptidase activities thus quantified were used without success to pursue the purification of candidate APP processing enzyme activities from human brain tissue. To date, no credible candidate protease(s) for either process have emerged, and the results of the various studies have been conflicting.
For example, the numerous available studies have proposed that the pathologic protease is: a lysosomal cathepsin, Cataldo et al., 1990, Proc. Natl. Acad. Sci USA 87:3861-3865; a calcium dependent serine protease, Abrahams et al., 1990, J. Neuropathol. Exp. Neurol., 49:333 (abstract); Calpain I, Siman et al., 1990, J. Neuroscience 10:2400-2411; a multicatalytic protease, Ishiura et al., 1989, FEBS. Lett. 257:388-392; or a chymotryptic-like serine protease, Nelson et al., 1990, J. Biol Chem. 265:3836-3843.
Similar inconsistencies have arisen in the efforts to identify the secretase, which has been claimed to be: a metallo-peptidase, McDermott et al., 1991, Biochem. Biophys. Res. Comm. 179:1148-1154; an acetylcholinesterase associated protease, Small et al., 1991, Biochemistry 30:10795-10799; or Cathepsin B, Tagawa et al., Biochem. Biophys. Res. Comm. 177:377-387.
The general lack of success of past and current efforts to identify the nature of the APP processing enzymes have stemmed from poor specificity of the assays employed, and from the complex heterogeneity of proteases associated with the cerebral tissue.
The present invention arose from efforts aimed at identifying the APP processing enzymes using specific assays based on the proteolytic degradation of recombinant APP. It has been discovered that such assays have utility in the diagnosis of AD by the unexpected finding of substantially different levels of APP degrading enzyme activity in samples of cerebrospinal fluid (hereinafter "CSF") obtained from AD patients compared with healthy controls.
The present invention is an in vitro assay for detecting AD-related differences in the levels of proteolytic enzyme activity specific for APP 695 in a body fluid derived from a patient. It has been unexpectedly discovered that CSF derived from AD patients contain no detectable levels of protease activity or significantly and consistently lower levels of protease activity than corresponding control samples of CSF derived from non-AD individuals. Thus, this assay is suitable for use as a diagnostic for AD in humans, and would provide means of early detection as required for more effective early therapeutic intervention.
Based on available knowledge and data prior to the present disclosure, the logical expectation is for relatively increased protease activity resulting in C-100 fragments for fluids continuous with the CNS from AD patients. The results disclosed herein show the opposite, CSF from non-AD subjects show relatively and consistently higher enzyme activity resulting in the C-100 fragments.
Presently, the only means for conclusive confirmation of clinical diagnosis is post mortem examination of the brains of AD diagnosed patients for the presence of cerebrovascular amyloid deposits, neuritic plaques and neurofibrillary tangles. By contrast, the present assay can be performed on body fluid samples derived from live patients, to quantify protease activity.
Other reports of biochemical differences between control and AD patients which may be of potential diagnostic utility are known including, the detection of dermal amyloid deposits in AD using radioactive iodine substituted derivatives (U.S. Pat. No. 5,039,511); elevated plasma alpha-l-anti chymotrypsin levels in AD (Matsubara et al., 1990, Ann. Neurol. 28:561-567); and the presence of immunochemical markers such as A68, a 68 kDa protein expressed by degenerating neurons and detectable with the monoclonal antibody ALZ-50 (Wolozin et al., 1986, Science, 323:648-650).
U.S. Pat. No. 4,874,694, describes a method of testing CSF for non-specific peptides susceptable to protein kinase C-phosphorylation. Protein kinase C activity deviating from norm is said to be an indicator for a wide variety of neurological or psychiatric disorders, without any particular specificity.
Thus, there is a need in the art for a convenient diagnostic method which can confirm clinical indications of AD prior to the death of the patient. The large qualitative differences in activities obtained between control and AD patients in the present invention provides for a reliable clinical diagnostic method. The differences depend on enzymatic activity which offers the potential for further signal amplification by increasing the time period of incubation of the enzyme with the APP substrate.
Additionally, the format of the presently disclosed assays affords the capacity to process reasonably large numbers of samples and yields good sensitivity due to the immunochemical method of detection. Furthermore, the simplicity of the assay allows for ready adaptation for routine use by lab technicians and yields consistent, reproducible results. These and other improvements are described hereinbelow.